Devices for the thermal treatment of samples or reaction mixtures in a controlled way are used in several fields of chemistry and biochemistry. For example, it is known that chemical reaction rates are proportional to temperature. Also, the working time or shelf life of a biological samples or laboratory reagents can be increased by keeping the substance at an optimal temperature. Since labor time as well as reagents are expensive) it is desirable to increase the throughput of production and analysis, while at the same time, to minimize the necessary reaction volumes. In general such devices or instruments have a thermal block made of e.g. metal, composite, ceramic or the like, that is in thermal contact with the sample under investigation so that the temperature of the sample is affected by the temperature of the thermal block.
Particularly, a strong need for systems capable of cycling a sample through a range of temperatures, i.e. thermal cyclers, became apparent with the advent of the Polymerase Chain Reaction (PCR), a technique which revolutionized the field of health care and molecular diagnostics.
PCR enables isolation of genomic material, sequencing and the detection of genetic diseases, recombinant DNA techniques, genetic fingerprinting and paternity testing. Viral DNA can likewise be detected by PCR and the amount of virus (“viral load”) in a patient can be quantified by PCR-based DNA quantitation techniques or quantitative PCR.
Because the amount of product produced by PCR roughly correlates to the amount of starting material, PCR can be used to estimate the amount of a given sequence that is present in a sample and because of the high sensitivity, virus detection may be possible soon after infection and even before the onset of disease symptoms, thus giving a significant lead in treatment. Quantitative PCR is also useful for determining gene expression levels. In cells, each gene is expressed through the production of messenger RNA (mRNA), which is then used to create a protein corresponding to the gene. The amount of mRNA in the cell for a given gene reflects how active that gene is. By using reverse transcription to produce DNA complementary to the mRNA (called cDNA) and subsequently using PCR to amplify these molecules, the amount of DNA produced for each gives a rough measure of the underlying expression for that gene.
Real-time PCR is a special form of quantitative PCR. By this technique it is possible to simultaneously amplify and quantify a specific part of a given DNA molecule. The DNA is quantified after or during each round of amplification. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that generate fluorescence at a certain point during the cycle.
PCR specificity and yield as well as throughput are directly related to the ability of the thermal-cycling system to rapidly and accurately arrive at and maintain reaction temperatures for an array of samples in parallel, e.g. in a multiwell plate in contact with a metal thermal block. Heating and cooling is normally achieved by means of temperature regulating units such as thermoelectric coolers (TECs) also called Peltier elements as well as a heat sink. One problem in the prior art is that differences in sample temperature may be generated by non-uniformity of temperature from place to place within the sample block. Temperature gradients may exist within the material of the block, causing some samples to have different temperatures than others at particular times in the cycle. Further, since there are delays in transferring heat from the sample block to the sample, those delays may differ across the sample block. These differences in temperature and delays in heat transfer, commonly referred to as well-to-well inhomogeneity, may cause the yield of the PCR process to differ from sample vial to sample vial. To perform the PCR process successfully and efficiently, and to enable quantitative PCR, these time delays and temperature errors must be minimized to the greatest extent possible.
One state of the art instrument currently available on the market, is the LightCycler® 480 Real-Time PCR System from Roche Diagnostics. This instrument reduces the problem above thanks to a special architecture of the thermal block unit, which comprises also a so-called Therma-Base™ unit, located beneath the Peltier elements, for improved heat transfer and distribution to all samples within a multiwell plate. The heat sink below the Therma-Base® unit features a maximized inner surface area to facilitate rapid heat absorption.
In U.S. Pat. No. 7,133,726B1, it is proposed instead to use a perimeter trench for the heat sink and a perimeter heater around the metal thermal block to reduce edge losses as well as a pin at the center of the assembly establishing a thermal path from the sample block to the heat sink in order to compensate for thermal gradients.
A problem in the state of the art is however represented by the inefficient control of the thermal block unit. Data measured within the thermal block unit, e.g. temperature values, are sent to a controller unit of an instrument and the instrument controls the thermal block unit. An instrument or thermal block test is typically carried out only when the instrument is turned on. One disadvantage is that only a limited number of data are processed, thus making it difficult to react promptly to errors and/or failures and/or any deviation from the normal or expected functioning of the temperature regulating units. Also, data transfer may be unreliable due to the possible influence of the electric connections, e.g. the electric resistance of the cables itself, cracks or line interruptions between thermal block unit and instrument.